Project summary Chronic HIV infection produces pathologic inflammation which drives disease progression and contributes to the development of serious co-morbid medical conditions, even in the setting of effective combination antiretroviral therapy (CART); translocation of bacterial products across the gastrointestinal (GI) mucosa is a major antigenic stimulus for this process. Our research focuses on how vagal dysfunction (VD), which occurs commonly as part of HIV-associated neuropathy, affects GI and immune function in HIV. Our prior work has shown that HIV+ individuals with VD have a high prevalence of small intestinal bacterial overgrowth (SIBO), and that SIBO is associated with elevation of the pro-inflammatory cytokine IL-6, which predicts morbidity and mortality in HIV. We have also demonstrated that treatment with the acetylcholinesterase inhibitor pyridostigmine, reduces indirect markers of bacterial translocation (sCD14) and the pro-inflammatory cytokine TNF?. The current proposal builds on this work, with the overarching goal of examining vagally-mediated GI mechanisms which could contribute to chronic inflammation in individuals with well-controlled HIV. Specifically we will seek to establish that small intestinal dysmotility and hypochlorhydria mediate the relationship between VD and SIBO, and to describe the changes in the GI microbiome in PLWH with SIBO. We will also determine whether VD is associated with elevations in IL-6 and TNF? independent of SIBO, establish to what degree the strength of these relationships depend on the presence of HIV infection, and whether they are reversible using pyridostigmine and/or non-invasive vagal nerve stimulation (nVNS). To achieve these aims, we will recruit 150 HIV+ participants who will undergo autonomic function tests for VD, hydrogen methane breath testing for SIBO, Wireless Motility Capsule (WMC, Smartpill) testing for GI regional transit times and pH measurements, oral and stool sample collection for characterization of the GI microbiome, and blood draw for quantification of inflammatory biomarkers. HIV-negative controls (N=100) will undergo the same assessments. Then a subset of 96 HIV+ participants will enter one of two eight-week interventional phases followed by repetition of the same testing battery: 1) double-blind treatment with pyridostigmine vs. placebo (N=86), or 2) open label treatment with non-invasive vagal nerve stimulation (N=10). These procedures will shed light on mechanisms linking VD to immune dysregulation in HIV, and provide support for potential therapies.